Molecular Characterization of Aromatic Rice Genotypes using Microsatellite Markers
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Date
2018
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Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur
Abstract
A study was conducted to examine the nature and extent of genetic differentiation and divergence among the aromatic rice genotypes using microsatellite markers.The materials were grown in petriplates for extraction of genomic DNA from the young seedlings and then targeted amplification of the genomic DNA was achieved by using a panel of eighteen microsatellite based primer pairs covering four chromosomes in the genome of rice. The statistical methods and parameters used for deriving inference were polymorphism per cent, polymorphism information content, discrimination coefficient, similarity coefficient, principal coordinate analysis and numerical taxonomic analysis of divergence.
Amplification was successfully achieved with all the microsatellite primers used in the present study. Appearance of bands at different positions on the gel revealed differential migration of amplified products due to differences in overall size of the products generated from targeted amplification of specific region of genome. The polymorphism among the entries was recognized on the basis of presence or absence of bands, in addition to variation in respect of number and position of bands.Altogether 123 allelic variants were detected among the eighteen aromatic rice entries with an average of 4.92 alleles per locus.The number of allelic variants per primer pair ranged from three in the case of E03.92.0 to ten in the case of RM 252, RM 256, RM 284 and RM 7356. A total of 76 shared and 47 unique allelic variants were generated amongst the entries.The primer pairs RM 7356, RM 252, RM 284, RM 223, RM 444, Aro7, E11.44.5, RM256 and RM 505 generated considerably greater percentage of unique alleles.
Appearance of more than one band in the same entry indicated the existence of the duplicated region in the genome. The primer pairs RM 223, RM 252, RM 444, RM 505, RM 252, RM 7049 and RM 7356 generated amplified products due to amplification of more than one locus. Considering the magnitude of polymorphic information content value in conjunction with number of alleles and polymorphism per cent, the primer pairs RM 256, RM 284, Aro7, RM 223, RM 252, RM 444, RM 7356appeared to be highly informative.Occurrence of null allele for a microsatellite locus associated with primer pairs RM 505 was noticed reflecting null allele in combination with one of the eighteen entries under evaluation.Relatively higher magnitude of discrimination coefficient was obtained for the primer pairs RM 256, RM 284, Aro7, RM 8264, RM 223, RM 252, RM 444, RM 7356, RM 7049, ARSSR-3 and RM 515, indicating their greater efficiency in respect of their ability to unambiguously discriminate the pair-wise combination of aromatic rice entries.
Microsatellite loci with tetra-nucleotide repeat motifs tended to detect greater number of alleles than the repeat loci withdi-nucleotide, tri-nucleotide and complex repeat motifs.Any relationship between the repeat number and the number of identified alleles was not observed in the present study.Simple sequence repeat loci with CT, GA and AT di-nucleotide repeat motifs tended to detect comparatively greater number of allelic variants. Ample genetic variation at molecular level amongst the aromatic rice entries under evaluation was inferred on the basis of the estimates of similarity coefficients. Considering the broad classification of entries, the entries appeared to bebasically divided into three groups, which were further divided into five clusters to allow the entries with relatively more similar pattern for markers to be clustered together. Principal coordinate analysis based two dimensional plots of the genetic profiles supported the results of hierarchical classification based grouping of entries.. Microsatellite marker based analysis revealed unique or entry specific allele which could be useful as molecular fingerprints in the unambiguous discrimination and identification of aromatic rice genotypes. The use of eighteen microsatellite markers in the analysis of aromatic rice entries exhibited a remarkably higher level of genetic polymorphism, which allowed unique genotyping of eighteen entries included in the analysis.
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